Method for detecting nucleosome adducts

ABSTRACT

The invention relates to a method for detecting and measuring the presence of nucleosome-protein adducts and the use of such measurements for the detection and diagnosis of disease. The invention also relates to a method of identifying nucleosome adduct biomarkers for the detection and diagnosis of disease and to biomarkers identified by said method.

FIELD OF THE INVENTION

The invention relates to a method for detecting and measuring thepresence of nucleosome-protein adducts and the use of such measurementsfor the detection and diagnosis of disease. The invention also relatesto a method of identifying nucleosome adduct biomarkers for thedetection and diagnosis of disease and to biomarkers identified by saidmethod.

BACKGROUND OF THE INVENTION

The human body comprises several hundred cell types. All of these celltypes contain the same genome but widely different phenotypes anddifferent functions in the body. This phenotypic diversity is due to thedifferential expression of the genome in different cell types. Thecontrol of differential gene expression is not entirely understood butthe basic mechanisms include gene regulation by a number ofinterconnected epigenetic signals associated with the gene, includingcontrol of the chromatin packing as euchromatin or heterochromatin,control of nucleosome positioning and nuclease accessible sites,methylation of DNA and variation in the structure of the nucleosomesaround which the DNA is wrapped.

The nucleosome is the basic unit of chromatin structure and consists ofa protein complex of eight highly conserved core histones (comprising ofa pair of each of the histones H2A, H2B, H3, and H4). Around thiscomplex is wrapped approximately 146 base pairs of DNA. Another histone,H1 or H5, acts as a linker and is involved in chromatin compaction. TheDNA is wound around consecutive nucleosomes in a structure often said toresemble “beads on a string” and this forms the basic structure of openor euchromatin. In compacted or heterochromatin this string is coiledand super coiled into a closed and complex structure (Herranz andEsteller, 2007).

Normal cell turnover in adult humans involves the creation by celldivision of some 10¹¹ cells daily and the death of a similar number,mainly by apoptosis. During the process of apoptosis chromatin is brokendown into mononucleosomes and oligonucleosomes which are released fromthe cells. Under normal condition these are removed and the level ofcirculating nucleosomes found in healthy subjects is low. Elevatedlevels are found in subjects with a variety of conditions including manycancers, auto-immune diseases, inflammatory conditions, stroke andmyocardial infarction (Holdenrieder & Stieber, 2009).

Mononucleosomes and oligonucleosomes can be detected by Enzyme-LinkedImmunoSorbant Assay (ELISA) and several methods have been reported(Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze et al,2003). These assays typically employ an anti-histone antibody (forexample anti-H2B, anti-H3 or anti-H1, H2A, H2B, H3 and H4) as captureantibody and an anti-DNA or anti-H2A-H2B-DNA complex antibody asdetection antibody. However, we have found that the results of theseassays do not agree with each other. Furthermore, although mostcirculating DNA in serum or plasma is reported to exist asmono-nucleosomes and oligo-nucleosomes (Holdenrieder et al, 2001),measured levels of nucleosomes and DNA in serum or plasma do not agreewell. The correlation coefficient between ELISA results for circulatingcell free nucleosomes levels and circulating DNA levels as measured byreal time PCR (Polymerase Chain Reaction) has been reported to ber=0.531 in serum and r=0.350 in plasma (Holdenrieder et al, 2005).

Nucleosome ELISA methods are used in cell culture, primarily as a methodto detect apoptosis (Salgame et al, 1997; Holdenrieder et al, 2001; vanNieuwenhuijze et al, 2003), and are also used for the measurement ofcirculating cell free nucleosomes in serum and plasma (Holdenrieder etal, 2001). Cell free serum and plasma nucleosome levels released intothe circulation by dying cells have been measured by ELISA methods instudies of a number of different cancers to evaluate their use as apotential biomarker (Holdenrieder et al, 2001). Mean circulatingnucleosome levels are reported to be high in most, but not all, cancersstudied. The highest circulating nucleosome levels were observed in lungcancer subjects. The lowest levels were observed in prostate cancer,which were within the normal range of healthy subjects. However,subjects with malignant tumours are reported to have serum nucleosomeconcentrations that varied considerably and some subjects with advancedtumour disease were found to have low circulating nucleosome levels,within the range measured for healthy subjects (Holdenrieder et al,2001). Because of this and the variety of non-cancer causes of raisednucleosome levels, circulating nucleosome levels are not used clinicallyas a biomarker of cancer (Holdenrieder and Stieber, 2009).

The structure of nucleosomes can vary by Post TranscriptionalModification (PTM) of histone proteins and by the inclusion of varianthistone proteins. PTM of histone proteins typically occurs on the tailsof the eight core histones and common modifications include acetylation,methylation or ubiquitination of lysine residues as well as methylationof arginine residues and phosphorylation of serine residues. Histonemodifications are known to be involved in epigenetic regulation of geneexpression (Herranz and Esteller, 2007). The structure of the nucleosomecan also vary by the inclusion of alternative histone isoforms orvariants which are different gene or splice products and have differentamino acid sequences. Histone variants can be classed into a number offamilies which are subdivided into individual types. The nucleotidesequences of a large number of histone variants are known and publiclyavailable for example in the National Human Genome Research InstituteNHGRI Histone DataBase (Mariño-Ramirez, L., Levine, K. M., Morales, M.,Zhang, S., Moreland, R. T., Baxevanis, A. D., and Landsman, D. TheHistone Database: an integrated resource for histones and histonefold-containing proteins. Database Vol. 2011. (Submitted) andhttp://genome.nhgri.nih.gov/histones/complete.shtml), the GenBank (NIHgenetic sequence) DataBase, the EMBL Nucleotide Sequence Database andthe DNA Data Bank of Japan (DDBJ).

Histone variant and histone modification patterns present in healthy anddiseased cells have been shown to differ in numerous (mostlyimmunohistochemical) studies (Herranz and Esteller, 2007). Onedisadvantage of immunohistochemical methods for clinical use is thattissue sample collection is invasive involving surgery or biopsy.

In addition to the epigenetic signaling mediated by nucleosome structureand position, control of gene expression in cells is also mediated bythe methylation status of DNA (Herranz and Esteller, 2007). It has beenknown in the art for some time that DNA may be methylated at the 5position of cytosine nucleotides to form 5-methylcytosine.

The involvement of DNA methylation in cancer was reported as early as1983 (Feinberg and Vogelstein, 1983). DNA methylation patterns observedin cancer cells differ from those of healthy cells. Repetitive elements,particularly around pericentromeric areas, are reported to behypomethylated in cancer relative to healthy cells but promoters ofspecific genes are reported to be hypermethylated in cancer. The balanceof these two effects is reported to result in global DNA hypomethylationin cancer cells (Rodriguez-Paredes & Esteller, 2011).

Hypermethylation of certain specific genes can be used as a diagnosticbiomarker for cancers. For example a method reported for detection ofhypermethylation of the Septin 9 gene by PCR amplification of DNAextracted from plasma was reported to detect 72% of colon cancers with afalse positive rate of 10% (Grutzmann et al, 2008). The DNA methylationstatus of specific genes or loci is usually detected by selectivebisulphite deamination of cytosine, but not 5-methylcytosine, to uracil,leading to a primary DNA sequence change that can be detected bysequencing or other means (Allen et al, 2004).

Global DNA hypomethylation is a hallmark of cancer cells (Esteller 2007and Hervouet et al, 2010). Global DNA methylation can be studied incells using immunohistochemistry techniques. Alternatively the DNA isextracted from the cells for analysis.

It has been known for many years that, in addition to nucleic acid andhistone proteins, chromatin comprises a large number of non-histoneproteins bound to its constituent DNA and/or histones (Yoshida andShimura, 1972). These chromatin associated proteins are of a widevariety of types and have a variety of functions including transcriptionfactors, transcription enhancement factors, transcription repressionfactors, histone modifying enzymes, DNA damage repair proteins and manymore. The study of chromatin bound proteins has been carried out largelyby Chromatin ImmunoPrecipitation (ChIP) methods. These methods are wellknown in the art but are complex, laborious and expensive.

In a typical ChIP method the cellular chromatin is cross-linked so thatall the protein and nucleic acid components are covalently attached toeach other. The chromatin is then sheared to form a preparation ofmononucleosomes and oligonucleosomes. An antibody to the protein ofinterest is added to the sheared chromatin to immunoprecipitate thosechromatin fragments containing the protein. The antibody is normallyattached to a solid phase (eg; plastic beads) to facilitate isolation ofthe chromatin complex containing the protein of interest. Thecross-linking is then reversed and the protein is removed by digestionwith a proteinase. The DNA associated with the chromatin complex isisolated and analysed to determine the DNA sequence, gene or locusassociated with the particular protein binding using any of a variety oftechniques including PCR followed by gel electrophoresis, DNA sequencing(ChIP-Seq) or DNA microarrays (ChIP-on-chip).

These ChIP methods reveal the DNA sequences associated with chromatinbound histone proteins. Derivatives of the ChIP method have beendeveloped to facilitate studies of the association of non-histoneproteins with histones and nucleosomes including for example HistoneAssociated Assays (Ricke and Bielinsky, 2005). Many proteins that bindto chromatin are involved in cancer and other disease mechanisms buttheir abundance in nucleosome adduct form in the circulation has notbeen previously investigated. Examples include the High Mobility GroupBox Protein 1 (HMGB1), the polycomb protein Enhancer of Zeste Homolog 2(EZH2) and the nuclear receptor group of proteins.

The High Mobility Group of proteins are a component of chromatin presentat about 3% of the weight of DNA or histones. They are structuralproteins that bind to nucleosomes without any known specificity for theunderlying DNA sequence (Gerlitz et al; 2009). HMGB1 is an architecturalchromosomal protein and a pro-inflammatory mediator. It is involved incell death, apoptosis and in numerous diseases including variousinflammatory and autoimmune conditions, sepsis, meningitis andneurodegeneration. Overexpression of HMGB1 is associated with all of thecentral hallmarks of cancer (Tang et al; 2010). HMGB1 is tightlyattached to the chromatin of apoptotic cells. Studies ofnucleosome-HMGB1 complexes have shown that these adducts are found inthe circulation of subjects suffering from the autoimmune diseaseSystemic Lupus Erythematosus (SLE) and that the adducts are involved inthe development of anti-nuclear antibodies which is a key feature ofSLE. Nucleosomes not attached to HMGB1 do not illicit an immuneresponse. The binding of HMGB1 to nucleosomes in these adducts wasdemonstrated by immunoprecipitation of nucleosomes with an antibodydirected to DNA or histones followed by Western Blot using an anti-HMGB1antibody to demonstrate the presence of HMGB1 in the immunoprecipitatednucleosomes (Urbonaviciute et al; 2008).

HMGB proteins interact with many other proteins known to affectchromatin function and chromatin complexes involving HMGB proteins plusadditional proteins have been shown to occur (Gerlitz et al; 2009).Thus, in addition to simple nucleosome-protein adducts,nucleosome-protein-complex adducts in which 2 or multiple proteins areassociated with nucleosomes occur in chromatin.

EZH2 is a member of the Polycomb-group (PcG) family that form multimericprotein complexes involved in maintaining the transcriptional repressivestate of genes. EZH2 is a histone modification enzyme (histone-lysineN-methyltransferase) that methylates the lysine 27 amino acid residue ofhistone 3 of nucleosomes. This histone modification is associated withchromatin condensation and gene silencing (Cao et al; 2002).

Nuclear receptors are molecules that regulate gene expression under thecontrol of hormones or ligands, for example the estrogen receptor (ER)regulates the expression of estrogen dependent genes. Many of theseproteins are involved in disease processes, for example ER is involvedin the progression of breast cancer and many breast cancer treatmentsare targeted to ER and/or to prevention of the interaction of ER withits ligand estradiol.

In addition to nucleosome-protein adducts that occur in the cell, thereare other nucleosome-protein adducts that may be formed after release ofnucleosomes from the cell following cell death. Such nucleosome adductsinclude the nucleosome-immunoglobulin adducts that are a key feature ofSLE.

We now report simple immunoassay methods for the direct estimation ofprotein-nucleosome adducts in biological samples. We have developedsimple methods for the detection of nucleosome bound EZH2, HMGB1 andseveral nuclear receptors and shown that such nucleosome adducts can bedetected in serum samples and that they have use as biomarkers indisease.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided the useof a nucleosome-protein adduct as a biomarker in blood for the diagnosisof cancer, autoimmune disease or inflammatory disease.

According to a second aspect of the invention there is provided a methodfor detecting the presence of a nucleosome-protein adduct in a samplewhich comprises the steps of:

-   -   (i) contacting the sample with a first binding agent which binds        to nucleosomes or a component thereof;    -   (ii) contacting the nucleosomes or sample with a second binding        agent which binds to a protein adducted to a nucleosome;    -   (iii) detecting or quantifying the binding of said second        binding agent to the adducted protein in the sample; and    -   (iv) using the presence or degree of such binding as a measure        of the presence of nucleosome adducts in the sample.

According to a third aspect of the invention there is provided a methodfor detecting the presence of a nucleosome adduct in a sample whichcomprises the steps of:

-   -   (i) contacting a sample with a first binding agent which binds        to a protein adducted to a nucleosome;    -   (ii) contacting the nucleosomes or sample with a second binding        agent which binds to nucleosomes or a component thereof;    -   (iii) detecting or quantifying the binding of said second        binding agent to nucleosomes or a component thereof in the        sample; and    -   (iv) using the presence or degree of such binding as a measure        of the presence of nucleosome adducts in the sample.

According to a further aspect of the invention there is provided amethod for detecting a nucleosome adduct in a cell which comprises thesteps of:

-   -   (i) isolating chromatin from a cell;    -   (ii) digesting, sonicating or otherwise breaking down the        chromatin to form mono-nucleosomes and/or oligo-nucleosomes; and    -   (iii) detecting or measuring the presence of the nucleosome        adduct according to an ELISA method of the invention described        in the above second or third aspects.

According to a further aspect of the invention there is provided amethod for detecting or diagnosing a disease status in an animal or ahuman subject which comprises the steps of:

-   -   (i) detecting or measuring a nucleosome adduct in a body fluid        of a subject; and    -   (ii) using the nucleosome adduct level detected to identify the        disease status of the subject.

According to a further aspect of the invention there is provided amethod for assessment of an animal or a human subject for suitabilityfor a medical treatment which comprises the steps of:

-   -   (i) detecting or measuring a nucleosome adduct in a body fluid        of the subject; and    -   (ii) using the nucleosome adduct level detected as a parameter        for selection of a suitable treatment for the subject.

According to a further aspect of the invention there is provided amethod for monitoring a treatment of an animal or a human subject whichcomprises the steps of:

-   -   (i) detecting or measuring a nucleosome adduct in a body fluid        of the subject;    -   (ii) repeating the detection or measurement of a nucleosome        adduct in a body fluid of the subject on one or more occasions;    -   (iii) using any changes in the nucleosome adduct level detected        as a parameter for any changes in the condition of the subject.

According to a further aspect of the invention there is provided amethod for identifying a nucleosome adduct biomarker for detecting ordiagnosing a disease status in an animal or a human subject whichcomprises the steps of:

-   -   (i) detecting or measuring a nucleosome adduct in a body fluid        of the subject;    -   (ii) detecting or measuring a nucleosome adduct in a body fluid        of a healthy subject or a control subject; and    -   (iii) using the difference between the levels detected in        diseased and control subjects to identify whether a nucleosome        adduct is useful as a biomarker for the disease status.

According to a further aspect of the invention there is provided abiomarker identified in accordance with methods defined herein.

According to a further aspect of the invention there is provided a kitfor the detection of a nucleosome adduct which comprises a ligand orbinder specific for the nucleosome adduct or component part thereof, ora structural/shape mimic of the DNA base, nucleotide or nucleoside orcomponent part thereof, together with instructions for use of the kit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: ELISA dose response curve for the detection of nucleosome EZH2adduct levels in digested chromatin extracted from Hela cells dilutedinto horse serum.

FIG. 2: Nucleosome-EZH2 adduct ELISA results for serum samples takenfrom 5 healthy subjects and 11 subjects with tumours.

FIG. 3: ELISA dose response curve for the detection of nucleosome-HMGB1adduct levels in digested chromatin extracted from Hela cells dilutedinto horse serum.

FIG. 4: Nucleosome-HMGB1 adduct ELISA results for serum samples takenfrom 5 healthy subjects and 11 subjects with tumours.

FIG. 5: Nucleosome-HMGB1 adduct ELISA results for serum samples takenfrom 31 healthy subjects and 74 subjects with (A) colon cancer, (B)breast cancer or (C) lung cancer.

FIG. 6: ELISA dose response curve for the detection ofnucleosome-Progesterone Receptor adduct levels in cell-free nucleosomesprepared by the method of *Holdenrieder et al; 2001.

FIG. 7: ELISA results for the detection of nucleosome-Androgen Receptoradduct levels in 2 prostate cancer cases and a cell-free nucleosomesample prepared by the method of *Holdenrieder et al; 2001.

FIG. 8: ELISA dose response curve for the detection ofnucleosome-Estrogen Receptor alpha (ERα) adduct levels in cell-freenucleosomes prepared by the method of *Holdenrieder et al; 2001.

FIG. 9: ELISA results for the detection of nucleosome-ERβ adduct levelsin digested MCF7 chromatin. The assay was carried out in two differentformats. In the first format the anti-nucleosome antibody was coated onthe wells and the anti-ERβ antibody was biotinylated. In the secondformat the anti-ERβ antibody was coated on the wells and theanti-nucleosome antibody was biotinylated.

FIG. 10: Nucleosome H2AZ-ERβ adduct ELISA results.

FIG. 11: Nucleosome-ERβ adduct ELISA results for serum samples takenfrom 12 healthy subjects and 16 subjects with tumours.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided the useof a nucleosome-protein adduct as a biomarker in blood for the diagnosisof cancer, autoimmune disease or inflammatory disease. In oneembodiment, the biomarker is used for the diagnosis of cancer. We haveshown that two such adducts containing HMGB1 and EZH2 are present in thecirculation of subjects with cancer but are not detected in thecirculation of healthy subjects.

It is well known in the art that cancers may be hormone dependent andrequire the presence of hormone for growth. It is also well known thatnuclear hormones function by nuclear localisation of the receptor boundhormone complex and binding to specific hormone response elements in thegenome. The expression of genes associated with the elements isregulated by binding of the receptor bound hormone complex to thegenomic response element. In one embodiment the invention provideshormone receptor-nucleosome adduct and hormone-hormonereceptor-nucleosome complex adduct biomarkers to characterise the tumourstatus of a subject. These adducts may be circulating adducts present inthe blood or in another body fluid or may be produced by the digestionof chromatin from a sample of tumour tissue.

It is well known in the art that nuclear hormone receptors regulate geneexpression under hormonal or ligand control. For example the estrogenreceptor functions by binding its substrate (the steroid hormoneestrogen) at the cell surface membrane. Binding is followed byinternalisation of the hormone-receptor complex and intra-nuclearlocalisation where the receptor binds to specific hormone responseelements in the genome. The specific gene sequence to which the estrogenreceptor binds is known as the Estrogen Response Element (ERE).Expression of genes associated with the ERE may be regulated by thereceptor and hence by the presence or level of estrogen in thecirculation of a subject. It is also well known in the art that growthof breast cancer is often under estrogen control and such cancer isoften termed estrogen dependent. As these tumours over express theEstrogen Receptor (ER) they are often termed ER+ tumours. The growth ofestrogen dependent tumours can be slowed or prevented by therapeuticinterventions aimed at prevention of estrogen binding to the estrogenreceptor and this is a common method of breast cancer treatment.Examples of such treatments include the drug Tamoxifen which acts as anantagonist for estrogen in estrogen dependent breast cancer andaromatase inhibitors which slow or prevent estrogen production. However,with time, cancers develop into estrogen independent tumours which willgrow even in the absence of estrogen stimulation and require differenttreatments. The diagnosis of estrogen dependent and independent tumoursis currently performed routinely by immunostaining of tumour biopsytissue to determine the abundance or otherwise of the estrogen receptorin tumour cells. Clinicians may need to retest the estrogen dependencyof a tumour repeatedly during the course of tumour treatment todetermine whether or not further estrogen dependent treatment isappropriate or whether the subject's treatment regime should be alteredto reflect the changing nature of the tumour as the disease progresses.Unfortunately current tests are suboptimal and require repeated painfulbiopsy on each occasion the test is performed. In one embodiment of theinvention the detection of estrogen receptor-nucleosome adducts in thecirculation of breast cancer patients is used as an indicator ofestrogen receptor binding to ERE in the nucleus of tumour cells as anindicator for estrogen dependency of a tumour to aid the selection ofappropriate treatment and for predictive prognostic information. Thismethod has the advantages that it is indicative of ERE-estrogen receptorbinding in the tumour, rather than a simple indicator of the presence orabundance of estrogen receptor, and that it may be repeated asfrequently as desired by a simple blood test without the need forbiopsy. We have developed simple ELISA methods for the detection andquantification of nucleosome-ER adducts containing both the ERα and ERβforms of the receptor. Surprisingly these adducts are present in thecirculation of cancer patients.

It will be clear to those skilled in the art that the same principle canbe applied to the detection of estrogen receptor-nucleosome adducts incell chromatin digests produced from the tumour tissue itself. Thismethod for assessing the estrogen dependency of a tumour is superior tocurrent methods because it is indicative of ERE-estrogen receptorbinding in the tumour, rather than a simple indicator of the presence orabundance of estrogen receptor.

In another embodiment of the invention the detection of the presence ofsteroid estrogen itself in an estrogen-estrogen receptor-nucleosomecomplex adduct either in the circulation, or in another body fluid, orin nucleosomes produced as a digest of chromatin from tumour tissue isused as an indicator of estrogen dependency status of a tumour.

It is reported that circulating nucleosomes are elevated inendometriosis (Holdenrieder et al; 2001) and, as endometriosis tissue isestrogen responsive, binding of the estrogen receptor in the chromatinof endometriosis cells may lead to estrogen receptor-nucleosome adductsor estrogen-estrogen receptor-nucleosome complex adducts in thecirculation. In a further embodiment of the invention estrogenreceptor-nucleosome adducts or estrogen-estrogen receptor-nucleosomecomplex adducts are detected in a body fluid as a biomarker for thepresence of an estrogen dependent gynaecological condition including forexample endometriosis.

In a similar manner to estrogen dependent breast cancer, the growth ofandrogen dependent prostate cancer requires, or is accelerated byandrogen. Androgen dependent prostate tumours are similarly treated bymethods that prevent androgen binding to the androgen receptor (AR).Androgen dependent prostate tumours may also develop to become androgenindependent and hence resistant to treatments including physical orchemical castration by drugs to prevent androgen binding to itsreceptor. The androgen dependency status of a tumour may be determinedby the level of androgen receptor binding to androgen response elements(ARE) in the genome and this may be determined by the analysis ofandrogen receptor-nucleosome adduct levels present in the circulation ofa subject or in chromatin digests from prostate tissue. Embodiments ofthe invention for this purpose include the detection of androgenreceptor-nucleosome adducts or androgen-androgen receptor-nucleosomecomplex adducts in the circulation or in a body fluid of a subject or innucleosomes produced by digestion of chromatin from tumour tissue of asubject. We have now developed simple ELISA methods for the detectionand quantification of nucleosome-AR adducts and demonstrated theirutility. We have also developed simple ELISA methods for the detectionand quantification of nucleosome-Progesterone Receptor adducts. Otherhormone dependent diseases may be addressed with similar embodiments ofthe method of the invention. Such embodiments include the detection ofother receptor-nucleosome adducts including for example glucocorticoidreceptor, thyroid hormone receptor and retinoic acid receptor-nucleosomeadducts for the detection of tumours including for example various typesof leukaemia involving the retinoic acid receptor.

According to a further aspect of the invention, the methods describedhereinbefore may be used to detect hormone-hormone receptor-nucleosomecomplex adducts. In one embodiment, the hormone-hormonereceptor-nucleosome complex adducts comprise a thyroxine-thyroid hormonereceptor-nucleosome complex adduct, a triiodothyronine-thyroid hormonereceptor-nucleosome complex adduct, a retinoic acid-retinoic acidreceptor-nucleosome complex adduct, an androgen-androgenreceptor-nucleosome complex adduct or an estrogen-estrogenreceptor-nucleosome complex adduct. This aspect of the invention has theadvantage of distinguishing hormone activated adducts as well as adductscontaining wild type or normal hormone receptor from hormone receptorwhich does not bind its ligand, for example due to mutation in thecourse of disease progression (for example in estrogen independentbreast cancer). This aspect of the invention may be carried out inmultiple ways. In one embodiment an antibody or other binder directed tobind to the hormone itself is used in place of the antibody directed tobind the hormone receptor.

In an alternative embodiment hormone is extracted from an antibodycaptured hormone-hormone receptor-nucleosome complex adduct andquantified by established methods for example immunoassay methods,spectrographic methods or chromatographic methods including highperformance liquid chromatography (HPLC), liquid chromatography followedby mass spectroscopy (LC/MS) or gas chromatography followed by massspectroscopy (GC/MS). For example, androgen-androgen receptor-nucleosomecomplex adduct is captured by immobilised antibodies directed to bind toan epitope present on the adduct (for example on the androgen receptoror on a nucleosome). The hormone is then extracted from the solid phasebound adduct into an organic solvent (for example; diethyl ether). Thesolvent is transferred, dried and the androgen is redissolved in assaybuffer and its concentration is measured (for example by competitiveimmunoassay). It will be clear to those skilled in the art that thisembodiment will have particular application for small molecule hormonessuch as steroid and thyroid hormones.

The present invention is aimed at detection of proteins which are boundto nucleosomes. This can be done by means of a double antibody ELISAtest in which one antibody is directed to bind nucleosomes and the otheris directed to bind to the protein bound to the nucleosome. However theantibody directed to bind to the nucleosome need not be directed to thewhole nucleosome complex but may be directed to a protein or nucleicacid component part of the nucleosome. In this embodiment of theinvention the antibody employed to bind to the nucleosome may bedirected to bind any component part of a nucleosome including, forexample to a particular histone, histone modification, histone variantor isoform or to a particular nucleotide or modified nucleotide. We haveshown that this design of assay works well using the example of using anantibody directed to bind to the histone variant H2AZ as a binder ofnucleosomes. It will be clear to those skilled in the art that thismethod has the additional advantage of selectively binding only thosenucleosomes which contain both the protein of interest in the adduct andH2AZ. This design provides a method for an assay to test for anycombination of adduct protein with any particular histone, histonemodification, histone variant, nucleotide, modified nucleotide or othernucleosome structure.

According to a second aspect of the invention there is provided a methodfor detecting the presence of a nucleosome-protein adduct in a samplewhich comprises the steps of:

(i) contacting the sample with a first binding agent which binds tonucleosomes or a component thereof;(ii) contacting the nucleosomes or sample with a second binding agentwhich binds to a protein adducted to a nucleosome;(iii) detecting or quantifying the binding of said second binding agentto the adducted protein in the sample; and(iv) using the presence or degree of such binding as a measure of thepresence of nucleosome adducts in the sample.

It will be clear to those skilled in the art that the binding agent tobe detected may be selected to be either the antibody directed to theadducted protein or to the nucleosome or a component part of thenucleosome.

According to a third aspect of the invention there is provided a methodfor detecting the presence of a nucleosome adduct in a sample whichcomprises the steps of:

(i) contacting a sample with a first binding agent which binds to aprotein adducted to a nucleosome;(ii) contacting the nucleosomes or sample with a second binding agentwhich binds to nucleosomes or a component thereof;(iii) detecting or quantifying the binding of said second binding agentto nucleosomes or a component thereof in the sample; and(iv) using the presence or degree of such binding as a measure of thepresence of nucleosome adducts in the sample.

In one embodiment, the nucleosome adduct includes a pro-inflammatoryprotein, a High Mobility Group Protein, a polycomb protein, a chromatinmodifying enzyme, a nuclear receptor or a hormone. In an alternativeembodiment, the nucleosome adduct includes a High Mobility GroupProtein, a polycomb protein, a chromatin modifying enzyme, a hormonereceptor or a hormone. In a further embodiment, the nucleosome adductincludes a chromatin modifying enzyme, a nuclear receptor or a hormone.In a further embodiment, the High Mobility Group Protein is HMGB1. Inone embodiment, when the biomarker is used for the diagnosis of cancer,the nucleosome-protein adduct includes a High Mobility Group Protein.

In one embodiment, the chromatin modifying enzyme is a histoneacetylation, deacetylation, methylation, demethylation phosphorylation,dephosphorylation ubiquitination, deubiquitination sumoylation,desumoylation or DNA methyltransferase enzyme. In an alternativeembodiment, the chromatin modifying enzyme is EZH2.

In one embodiment, when the nucleosome-protein adduct includes a nuclearreceptor, said nuclear receptor is the estrogen receptor, androgenreceptor, progesterone receptor, thyroid hormone receptor,glucocorticoid receptor or retinoic acid receptor. In an alternativeembodiment, when the nucleosome-protein adduct includes a nuclearreceptor, said nuclear receptor is the estrogen receptor, androgenreceptor or retinoic acid receptor.

In one embodiment, when the nucleosome-protein adduct includes ahormone, said hormone is a thyroid hormone, a glucocorticoid hormone ora steroid hormone including an estrogen, an androgen, a progestogen, acorticosteroid or retinoic acid. In an alternative embodiment, when thenucleosome-protein adduct includes a hormone, said hormone is a steroidhormone including an estrogen, an androgen, a corticosteroid or retinoicacid.

In one embodiment, when the nucleosome-protein adduct includes a hormonereceptor, said hormone receptor is the estrogen receptor, androgenreceptor, progesterone receptor, thyroid hormone receptor or retinoicacid receptor.

We have shown that the method can be performed using an antibodydirected to the nucleosome itself in combination with an antibodydirected to bind to the protein adducted to the nucleosome or using anantibody directed to a component of a nucleosome, again in combinationwith an antibody directed to bind to the protein adducted to thenucleosome. In one embodiment, the nucleosome or nucleosome componentantibody or binder is directed to bind a particular epigeneticnucleosome epitope; for example any histone variant (eg; H2AZ), anyhistone modification (eg; trimethyl H3K9) or any nucleotide or modifiednucleotide (eg; 5-methylcytosine). In an alternative embodiment, thenucleosome or nucleosome component binder is directed to bind to aparticular epigenetic signal structure such that only a particularsubset of nucleosome adducts containing said epigenetic signal structureare detected.

In one embodiment, the binding agent used is an antibody, an antibodyfragment or an aptamer. In a further embodiment, the binding agent usedis an antibody.

In one embodiment, the sample is a biological fluid. In a furtherembodiment, the sample is blood or serum or plasma. It will be clear tothose skilled in the art that the detection of nucleosome adducts in abody fluid has the advantage of being a minimally invasive method thatdoes not require biopsy.

In some cases however, it may be preferable to assess the nucleosomeadduct status of a cell directly by producing nucleosomes from that celland analyzing the nucleosomes for the presence of particular nucleosomeadducts.

According to a further aspect of the invention there is provided amethod for detecting a nucleosome adduct in a cell which comprises thesteps of:

-   -   (i) isolating chromatin from a cell;    -   (ii) digesting, sonicating or otherwise breaking down the        chromatin to form mono-nucleosomes and/or oligo-nucleosomes; and    -   (iii) detecting or measuring the presence of the nucleosome        adduct according to an ELISA method of the invention described        in any of the above second to sixth aspects.

According to a further aspect of the invention there is provided amethod for detecting or diagnosing a disease status in an animal or ahuman subject which comprises the steps of:

-   -   (i) detecting or measuring a nucleosome adduct in a body fluid        of a subject; and    -   (ii) using the nucleosome adduct level detected to identify the        disease status of the subject.

In one embodiment of the invention the presence of a nucleosome adductin a sample is used to determine the optimal treatment regime for asubject in need of such treatment. One example of such an embodiment isthe detection of a nuclear hormone receptor-nucleosome adduct or ahormone-hormone receptor-nucleosome complex adduct for assessment of thehormone dependency of a tumour.

According to a further aspect of the invention there is provided amethod for assessment of an animal or a human subject for suitabilityfor a medical treatment which comprises the steps of:

-   -   (i) detecting or measuring a nucleosome adduct in a body fluid        of the subject; and    -   (ii) using the nucleosome adduct level detected as a parameter        for selection of a suitable treatment for the subject.

According to a further aspect of the invention there is provided amethod for monitoring a treatment of an animal or a human subject whichcomprises the steps of:

-   -   (i) detecting or measuring a nucleosome adduct in a body fluid        of the subject;    -   (ii) repeating the detection or measurement of a nucleosome        adduct in a body fluid of the subject on one or more occasions;    -   (iii) using any changes in the nucleosome adduct level detected        as a parameter for any changes in the condition of the subject.

In one embodiment, the nucleosome adduct is detected or measured as oneof a panel of measurements.

According to a further aspect of the invention there is provided amethod for detecting or measuring a nucleosome adduct, either alone oras part of a panel of measurements, for the purposes of detecting ordiagnosing a disease status, or for assessment of an animal or a humansubject for suitability for a medical treatment, or for monitoring atreatment of an animal or a human subject, for use in subjects withactual or suspected cancer, benign tumours, inflammatory disease,autoimmune disease, endometriosis, infectious disease, sepsis, stroke ormyocardial infarction.

According to a further aspect of the invention there is provided amethod for identifying a nucleosome adduct biomarker for detecting ordiagnosing a disease status in an animal or a human subject whichcomprises the steps of:

-   -   (i) detecting or measuring a nucleosome adduct in a body fluid        of the subject;    -   (ii) detecting or measuring a nucleosome adduct in a body fluid        of a healthy subject or a control subject; and    -   (iii) using the difference between the levels detected in        diseased and control subjects to identify whether a nucleosome        adduct is useful as a biomarker for the disease status.

According to a further aspect of the invention there is provided a kitfor the detection of a nucleosome adduct which comprises a ligand orbinder specific for the nucleosome adduct or component part thereof, ora structural/shape mimic of the DNA base, nucleotide or nucleoside orcomponent part thereof, together with instructions for use of the kit.

In addition to histone and nucleic acid components, chromatin is knownto contain a wide variety of proteins that perform a wide range offunctions. We selected HMGB1, EZH2 and several nuclear receptors asexamples of these proteins and have developed simple ELISA methods forthe detection of mononucleosome and oligonucleosome adducts of theseproteins. We performed these ELISA methods directly on serum samplestaken from healthy and diseased subjects and the methods require nosample extraction or other sample pre-treatment. Surprisingly we haveshown that these nucleosome adducts can be detected in the serum ofcancer subjects and that nucleosome adduct ELISA assays are useful inthe detection and diagnosis of disease states.

HMGB1 is a damage associated molecular pattern (DAMP) protein associatedwith cell death, apoptosis and numerous diseases including variousinflammatory and autoimmune conditions, sepsis, meningitis,neurodegeneration, SLE and cancer (Tang et al; 2010). Elevatedexpression of HMGB1 occurs in many cancers and is thought to beassociated with invasion and metastases (Sims et al, 2010). Elevatedlevels of HMGB1 also occur in the blood of cancer patients as well as ina variety of other conditions (Stoetzer et al, 2012). Circulating HMGB1can be measured by ELISA but such measurements are not used in routineclinical practice because circulating HMGB1 occurs in bound and freeforms and the Western immunoblot methods currently available todistinguish these are not suitable for routine use. Therefore there is aneed for a reliable method to distinguish between free HMGB1 and HMGB1complexes (Urbonaviciute and Voll, 2011). An important class ofcirculating HMGB1 complexes is HMGB1-nucleosome adducts and oneembodiment of the present invention is directed to the detection ofHMGB1-nucleosome adducts and other HMG-nucleosome adducts. We have shownthat HMG-nucleosome adducts can be measured in the blood of cancerpatients using a rapid and simple ELISA method.

HMGB1 is tightly attached to the chromatin of apoptotic cells. Studiesof nucleosome-HMGB1 complexes have shown that these adducts are found inthe circulation of subjects suffering from the autoimmune disease SLEand that the adducts are involved in the development of anti-nuclearantibodies which is a key feature of SLE. The presence of these adductsin the circulation has not been used for clinical diagnostic purposesbecause the Western blot methods used for their detection are expensive,slow and laborious and not suitable for routine clinical use. Thepresent invention overcomes these shortcomings.

EZH2 is a chromatin modification enzyme (histone-lysineN-methyltransferase) that methylates the lysine 27 amino acid residue ofhistone 3 of nucleosomes leading to chromatin condensation and genesilencing (Cao et al; 2002). This protein is known to bind chromatin inthe nucleus of living cells. Surprisingly we have shown that EZH2remains bound to nucleosomes after cell death and mononucleosome-EZH2and oligonucleosome-EZH2 adducts can be detected in the serum of cancersubjects using the novel ELISA methods of the present invention.

It is known that chromatin modifying enzymes are involved in cancer(Fullgrabe et al, 2011) and inhibition of the activity of these enzymesthrough the use of targeted drugs is a major form of cancer therapy.These drugs include for example and without limitation HistoneDeacetylation Complex inhibitors (HDACi), Histone Methyl Transferaseinhibitors (HMTi) and DNA Methyl Transferase inhibitors (DNMTi). Whilstthe presence of HMGB1 adducts in the circulation is known to bepathological and associated with anti-nuclear antibodies, the findingthat chromatin modifying enzyme-nucleosome adducts are present in thecirculation has not previously been reported. Assays for chromatinmodifying enzyme-nucleosome adducts have multiple uses in cancerincluding for example in the assessment of cancer disease states and inthe determination of the efficacy of chromatin modifying enzymeinhibitor drugs, for example to determine if the level of circulatingchromatin modifying enzyme-nucleosome adduct is altered by treatmentwith particular drugs. The method of the invention may be used todetermine circulating chromatin modifying enzyme-nucleosome adductlevels for a wide variety of disease diagnostic purposes includingdisease detection, monitoring, prognosis, differential diagnosis andchoice of treatment regimes. We have shown that nucleosome adductscontaining the HMT enzyme EZH2 can be detected in the circulation ofcancer patients. It will be clear to those skilled in the art that themethod of the invention may be applied to other chromatin modifyingenzymes including the before mentioned HDAC and DNMT enzymes as well asmany other enzymes including for example enzymes for histoneacetylation, demethylation, phosphorylation, dephosphorylation,ubiquitination, deubiquitination, sumoylation and desumoylation.

Nuclear receptors exert their gene regulatory effects in the nucleusunder ligand hormone control. Examples include the steroid hormonereceptors, thyroid receptor, glucocorticoid receptor and retinoic acidand vitamin D receptor. These receptors are involved in a variety ofcancer and other disease mechanisms. Some examples include theinvolvement of the Retinoic Acid Receptor (RAR) in leukaemia, theEstrogen Receptor (ER) in breast cancer and endometriosis, the AndrogenReceptor (AR) in prostate cancer and the Thyroid Hormone Receptor inthyroid disease and cancer.

Surprisingly we have shown that nuclear receptor-nucleosome adducts canbe detected in the circulation of cancer patients.

Thus all of the intra-cellular chromatin associated proteins we chose tostudy can be found in the serum of cancer patients in the form ofnucleosome adducts. These findings indicate that such adducts may not beunusual and that many such intra-cellular nucleosome protein adducts,involving many different chromatin associated proteins, may retain theirintegrity following cell death and be amenable to detection in the serumof cancer, auto-immune and inflammatory disease patients by the methodof the present invention.

We have used an anti-histone antibody as capture antibody for theseassays in combination with an appropriate specific anti-chromatinprotein (anti-HMGB1, anti-EZH2 or anti-nuclear receptor) antibody. Wehave used the assays to show that nucleosome adducts containing specificproteins can be measured in blood samples taken from subjects withcancer and are discriminating for use as non-invasive or minimallyinvasive biomarkers. The nucleosome-adduct levels detected in serumsamples taken from diseased subjects differed from those detected inserum samples from healthy subjects.

We measured the levels of circulating cell free nucleosome-HMGB1 andnucleosome-EZH2 adducts in blood samples taken from 3 subjects withcolon cancer, 6 subjects with lung cancer and 2 subjects with pancreaticcancer and compared these with the levels present in blood samples from5 healthy subjects as well as with an artificially produced preparationof serum nucleosomes from healthy subjects prepared as described in theliterature (*Holdenrieder et al, 2001) and with a commercially availablepreparation of nucleosomes prepared by digestion of chromatin extractedfrom Hela cells.

Normal ranges were calculated from the results for the 5 healthysubjects (mean result ±2 standard deviations of the mean) for thenucleosome-HMBG1 and nucleosome-EZH2 adducts and the results for cancersubjects were examined to see if they fall within, or outside of, therespective normal range. The data show that 2 of 3 colon cancer samples,4 of 6 lung cancer samples and 1 of 2 pancreatic cancer samples hadelevated nucleosome-HMBG1 adduct levels and similarly that 2 of 3 coloncancer samples, 4 of 6 lung cancer samples and 1 of 2 pancreatic cancersamples had elevated nucleosome-EZH2 adduct levels (Optical Densityresults higher than the top of the normal range).

We have similarly measured the levels of nuclear receptor-nucleosomeadducts in healthy and diseased patients and shown that these arepresent in the serum of cancer patients.

Proteins that bind to chromatin include, without limitation, nuclearreceptors, the High Mobility Group proteins (such as HMGB1), polycombproteins, chromatin modification enzymes (such as EZH2), DNAmodification enzymes, nuclear receptors, transcription factors,architectural or structural proteins, transcription enhancement factors,transcription repression factors, replication proteins, DNA damagerepair proteins and any other proteins involved in the control of geneexpression, chromatin packing or replication.

Nucleosome adducts can also occur due to binding of nucleosomes presentin a biological fluid after cell death. An example of such an adductwould be a nucleosome-antibody adduct formed an autoimmune disease suchas SLE.

Thus in one embodiment of the invention there is provided a method fordetecting or measuring the presence of a nucleosome-protein complex oradduct. The nucleosome adducts to be measured may be of any originincluding, without limitation, naturally occurring nucleosome adductspresent in biological fluids as a consequence of a healthy or diseasedcondition or nucleosome adducts may be produced by the digestion ofchromatin extracted from cells, or they may be produced by inducedapoptosis or necrosis of cells (for example by the method of*Holdenrieder et al; 2001). Surprisingly, we have shown that nucleosomeadducts occur in all these situations and can be detected by the methodof the invention.

In another embodiment of the invention there is provided a method fordetecting or measuring the presence of a nucleosome-protein adduct in abiological fluid.

In a further embodiment of the invention there is provided a method fordetecting or diagnosing the presence, type, recurrence or severity of adisease or assessing optimal drug or other treatment options by testinga subject sample for the presence or level of one or morenucleosome-protein complexes or adducts.

In a further embodiment of the invention there is provided a method fordetecting or diagnosing the presence, type, recurrence or severity of adisease or assessing optimal drug or other treatment options by testinga sample taken from a subject for the presence or level of anucleosome-protein complex or adduct as part of a panel of tests. AnELISA method for the detection of cell free nucleosomes containingdifferent histone modifications has been reported (Bawden et al; 2005).

Thus, such a panel of tests may consist, for example, of two or moremeasurements of nucleosomes containing different nucleosome epitopes;including without limitation different adducts and/or histonemodifications and/or histone variants and/or modified nucleotides and/ormeasurements of nucleosomes per se, or any combination or ratio of anyof these and any other nucleosome epitopes, as an indicator of thehealth or disease status of a subject.

We conclude that the method of the present invention is a successfulmethod for the detection and measurement of nucleosome adductscontaining particular proteins, and that this method is a superiormethod for the detection of nucleosome adducts than the methods of thecurrent art. The method is rapid, low cost and suitable for use incomplex biological media and fluids including blood and its derivatives.We have demonstrated that the method of the current invention can beused to detect nucleosome adducts in blood, and that this may be used asa biomarker for cancer. It will be clear to those skilled in the artthat a biomarker present in the blood has value for a broad range ofdiagnostic and disease screening purposes for cancer and other diseaseswhich are associated with elevated circulating nucleosomes (Holdenriederet al, 2001).

According to one aspect of the invention there is provided a doubleantibody, immunometric or sandwich immunoassay method for detecting andmeasuring cell free nucleosome adducts in a sample. One embodiment ofthis aspect is an immunoassay which comprises the steps of:

-   -   (i) contacting a sample which may contain nucleosome adducts        with a first antibody or other binder which binds to nucleosomes        or a component thereof;    -   (ii) contacting the nucleosomes or sample with a second antibody        or other binder which binds to a protein that may be present as        a nucleosome-protein adduct;    -   (iii) detecting and/or quantifying the binding of said second        antibody or other binder to a nucleosome-protein adduct in the        sample; and    -   (iv) using the presence or degree of such binding as a measure        of the presence of a nucleosome-protein adduct in the sample.

According to another aspect of the invention there is provided a methodfor detecting and measuring cell free nucleosome adducts in a sample byan immunometric immunoassay which comprises the steps of:

-   -   (i) contacting a sample which may contain nucleosome adducts        containing a particular protein with a first antibody or other        binder which binds to the protein of interest;    -   (ii) contacting the nucleosomes or sample with a second antibody        or other binder which binds to nucleosomes or a component        thereof;    -   (iii) detecting and/or quantifying the binding of said second        antibody or other binder to nucleosomes sample with a second        antibody or other binder which binds to nucleosomes or a        component thereof in the sample; and    -   (iv) using the presence or degree of such binding as a measure        of the presence of a nucleosome adduct in the sample.

It will be clear to those skilled in the art that the antibody or otherbinder used to bind nucleosomes or a component thereof in stage (i) ofthe first aspect above and stage (ii) of the second aspect above may bean antibody (or other binder) directed against intact nucleosomes oragainst any component part of a nucleosome including without limitationagainst a histone, a histone variant, a histone modification, anucleotide, a modified nucleotide or other part of the DNA component ofa nucleosome. Thus in a further aspect of the invention there isprovided a method for detecting (only) those nucleosome-protein adductswhich additionally contain another feature to which this binder isdirected including without limitation a particular histone modification,histone variant or nucleotide. An advantage of this design is that thenucleosome component epitope and adducted protein epitope of the assaycan be selected to be epitopes whose levels both differ greatly inhealthy or diseased patients, or other patient status underinvestigation. Thus is likely to reduce the proportion of nucleosomesdetected by the assay but to increase the clinical selectivity orspecificity of the assay.

We have performed this design of assay using an antibody directed to thenucleosome component H2AZ as the anti-nucleosome antibody in conjunctionwith anti-EZH2 antibodies and shown that nucleosome-EZH2 adductsspecifically associated with H2AZ can be detected by such assays andthat these assays can be used to discriminate between samples taken fromhealthy and diseased subjects.

In a further aspect of the invention the nucleosome adduct to bedetected may contain more than one protein. Further proteins in anadduct may be directly or indirectly bound to the nucleosome. Forexample a nucleosome may be bound to a HMGB protein and additionally toa further protein or proteins. The further protein(s) may be directlybound to the nucleosome or may be bound to the HMGB protein, and henceindirectly to the nucleosome. Nucleosome adducts may contain largeprotein complexes consisting of multiple protein components where thebinding of a particular protein in the complex adduct to the nucleosomemay be through multiple intermediary binding connections. It will beclear to those skilled in the art that a protein bound to a nucleosomein a nucleosome adduct, either directly or indirectly, may be detectedby a method of the present invention.

It will be clear to those skilled in the art that the methods of theinvention described include a variety of embodiments including biosensortype assays and label-free assays of the type marketed for example byForteBio Incorporated of USA.

According to a further aspect of the invention there is provided amethod for detecting the proportion of nucleosomes that comprises aparticular nucleosome adduct in a sample comprising the steps of:

-   -   (i) detecting or measuring the level of nucleosomes in a sample;    -   (ii) detecting or measuring the level of a nucleosome adduct        according to a method of the current invention; and    -   (iii) using the two measurements to determine the proportion of        nucleosomes that comprises the nucleotide adduct.

We have shown that the detection and measurement of nucleosome adductsin the blood taken from subjects can be used as a diagnostic method toidentify subjects with cancer and to differentiate them from healthysubjects. According to a further aspect of the invention there isprovided a method for detecting or diagnosing the presence of a diseaseby measuring or detecting the presence and/or the level or concentrationof cell free nucleosome adducts in a body fluid, and using the detectedlevel as a biomarker of the disease status of a subject including,without limitation, a clinical diagnosis of a disease, a differentialdiagnosis of disease type or subtype, or a disease prognosis, or adisease relapse, or a diagnosis of subject susceptibility to treatmentregimens. It will be appreciated by those skilled in the art that bodyfluids used for diagnostic testing include without limitation blood,serum, plasma, urine, cerebrospinal fluid and other fluids. In apreferred embodiment the body fluid selected as the sample is blood,serum or plasma. The assay response, level, concentration or quantity ofa nucleosome adduct in a body fluid may be expressed in absolute termsor relative terms, for example without limitation as a proportion of thetotal nucleosome level present or as a ratio to the level of nucleosomescontaining another nucleosome structure such as a histone modificationor to the level of total DNA.

In one embodiment of the invention the nucleosome adduct measurement isused as a member of a diagnostic panel of tests or measurements for thedetection or diagnosis of the disease status of a subject including,without limitation, a clinical diagnosis of a disease, a differentialdiagnosis of disease type or subtype, or a disease prognosis, or adisease relapse, or a diagnosis of subject susceptibility to treatmentregimens

According to another aspect of the invention there is provided a methodfor detecting or measuring the presence and/or the level of chromatinbinding of a protein in a cell which comprises the steps of:

-   -   (i) isolating chromatin from a cell;    -   (ii) breaking down the chromatin to form mono-nucleosomes and/or        oligo-nucleosomes; and    -   (iii) detecting or measuring the presence of a nucleosome adduct        in the mono-nucleosomes and/or oligo-nucleosomes by means of an        immunoassay method of the invention.

Methods for producing mono-nucleosomes and/or oligo-nucleosomes fromchromatin are well known in the art and include enzyme digestion andsonication (Dai et al, 2011). We have demonstrated this aspect fornucleosomes produced from Hela and from MCF7 cells.

It will be clear to those skilled in the art that the terms antibody,binder or ligand in regard to any aspect of the invention is notlimiting but intended to include antibody fragments, aptamers or anybinder capable of binding to particular molecules or entities and thatany suitable binder can be used in the method of the invention. It willalso be clear that the term nucleosomes is intended to includemononucleosomes and oligonucleosomes and any such chromatin fragmentsthat can be analysed in fluid media.

According to another aspect of the invention there is provided a kit fordetecting or measuring nucleosome adducts which comprises a ligand orbinder specific for the nucleosome adduct or a component part thereof,or a structural/shape mimic of the nucleosome adduct or component partthereof, together with instructions for use of the kit in accordancewith any of the methods defined herein.

According to another aspect of the invention there is provided a methodfor identifying a nucleosome adduct biomarker for detecting ordiagnosing disease status in animals or humans which comprises the stepsof:

-   -   (i) detecting or measuring the level of a cell free nucleosome        adduct in a body fluid of diseased subjects;    -   (ii) detecting or measuring the level of a cell free nucleosome        adduct in a body fluid of control subjects; and    -   (iii) using the difference between the levels detected in        diseased and control subjects to identify whether a nucleosome        adduct is useful as a biomarker for that disease.

It will be clear to those skilled in the art that the control subjectsmay be selected on a variety of basis which may include, for example,subjects known to be free of the disease or may be subjects with adifferent disease (for example; for the investigation of differentialdiagnosis).

According to a further aspect of the invention there is provided amethod for identifying a nucleosome adduct biomarker for assessing theprognosis of a diseased animal or human subject which comprises thesteps of:

-   -   (i) detecting or measuring the level of a cell free nucleosome        adduct in a body fluid of diseased subjects; and    -   (ii) correlating the level of cell free nucleosome adduct        detected in a body fluid of diseased subjects with the disease        outcome of the subjects.

According to a further aspect of the invention there is provided amethod for identifying a nucleosome adduct biomarker to be used for theselection of a treatment regimen for a diseased animal or human subjectin need of treatment which comprises the steps of:

-   -   (i) detecting or measuring the level of a cell free nucleosome        adduct in a body fluid of diseased subjects; and    -   (ii) correlating the level of cell free nucleosome adduct        detected in a body fluid of diseased subjects with the observed        efficacy of a treatment regimen in those subjects.

According to a further aspect of the invention there is provided amethod for identifying a nucleosome adduct biomarker to be used formonitoring the treatment of a diseased animal or human subject whichcomprises the steps of:

-   -   (i) detecting or measuring the level of a cell free nucleosome        adduct in a body fluid of a diseased subject;    -   (ii) repeating said detection or measurement on one or more        occasions during the disease progression of the subject; and    -   (iii) correlating the level of cell free nucleosome adduct        detected in a body fluid of a diseased subject with the disease        progression in the subject.

According to a further aspect of the invention, there is provided abiomarker identified by the method as defined herein.

A further aspect of the invention provides ligands or binders, such asnaturally occurring or chemically synthesised compounds, capable ofspecific binding to the biomarker. A ligand or binder according to theinvention may comprise a peptide, an antibody or a fragment thereof, ora synthetic ligand such as a plastic antibody, or an aptamer oroligonucleotide, capable of specific binding to the biomarker. Theantibody can be a monoclonal antibody or a fragment thereof capable ofspecific binding to the biomarker. A ligand according to the inventionmay be labeled with a detectable marker, such as a luminescent,fluorescent, enzyme or radioactive marker; alternatively or additionallya ligand according to the invention may be labelled with an affinitytag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag.Alternatively ligand binding may be determined using a label-freetechnology for example that of ForteBio Inc.

A biosensor according to the invention may comprise the biomarker or astructural/shape mimic thereof capable of specific binding to anantibody against the biomarker. Also provided is an array comprising aligand or mimic as described herein.

Also provided by the invention is the use of one or more ligands asdescribed herein, which may be naturally occurring or chemicallysynthesised, and is suitably a peptide, antibody or fragment thereof,aptamer or oligonucleotide, or the use of a biosensor of the invention,or an array of the invention, or a kit of the invention to detect and/orquantify the biomarker. In these uses, the detection and/orquantification can be performed on a biological sample as definedherein.

Diagnostic or monitoring kits are provided for performing methods of theinvention. Such kits will suitably comprise a ligand according to theinvention, for detection and/or quantification of the biomarker, and/ora biosensor, and/or an array as described herein, optionally togetherwith instructions for use of the kit.

A further aspect of the invention is a kit for detecting the presence ofa disease state, comprising a biosensor capable of detecting and/orquantifying one or more of the biomarkers as defined herein.

Biomarkers for detecting the presence of a disease are essential targetsfor discovery of novel targets and drug molecules that retard or haltprogression of the disorder. As the level of the biomarker is indicativeof disorder and of drug response, the biomarker is useful foridentification of novel therapeutic compounds in in vitro and/or in vivoassays. Biomarkers of the invention can be employed in methods forscreening for compounds that modulate the activity of the biomarker.

Thus, in a further aspect of the invention, there is provided the use ofa binder or ligand, as described, which can be a peptide, antibody orfragment thereof or aptamer or oligonucleotide according to theinvention; or the use of a biosensor according to the invention, or anarray according to the invention; or a kit according to the invention,to identify a substance capable of promoting and/or of suppressing thegeneration of the biomarker.

Also there is provided a method of identifying a substance capable ofpromoting or suppressing the generation of the biomarker in a subject,comprising administering a test substance to a subject animal anddetecting and/or quantifying the level of the biomarker present in atest sample from the subject.

The term “biomarker” means a distinctive biological or biologicallyderived indicator of a process, event, or condition. Biomarkers can beused in methods of diagnosis, e.g. clinical screening, and prognosisassessment and in monitoring the results of therapy, identifyingsubjects most likely to respond to a particular therapeutic treatment,drug screening and development. Biomarkers and uses thereof are valuablefor identification of new drug treatments and for discovery of newtargets for drug treatment.

The terms “detecting” and “diagnosing” as used herein encompassidentification, confirmation, and/or characterisation of a diseasestate. Methods of detecting, monitoring and of diagnosis according tothe invention are useful to confirm the existence of a disease, tomonitor development of the disease by assessing onset and progression,or to assess amelioration or regression of the disease. Methods ofdetecting, monitoring and of diagnosis are also useful in methods forassessment of clinical screening, prognosis, choice of therapy,evaluation of therapeutic benefit, i.e. for drug screening and drugdevelopment.

Efficient diagnosis and monitoring methods provide very powerful“subject solutions” with the potential for improved prognosis, byestablishing the correct diagnosis, allowing rapid identification of themost appropriate treatment (thus lessening unnecessary exposure toharmful drug side effects), and reducing relapse rates.

In one embodiment, said biomarker is released from the cells of atumour. Thus, according to a further aspect of the invention there isprovided a method for the detection of a tumour growth which comprisesthe steps of (i) measuring a biomarker in a biological sample that isassociated with or released from the cells of a tumour and (ii)demonstrating that the level of said biomarker is associated with thesize, stage, aggressiveness or dissemination of the tumour.

It is known that increased cell turnover, cell death and apoptosis leadto increased circulatory levels of cell free nucleosomes (Holdenriederet al, 2001). Circulating cell free nucleosomes level is a non-specificindicator and occurs in a variety of conditions including inflammatorydiseases, a large variety of benign and malignant conditions, autoimmunediseases, as well as following trauma or ischaemia (Holdenrieder et al2001). It will be clear to those skilled in the art that the inventionwill have application in a variety of disease areas where circulatingnucleosomes have been found in subjects. These include, withoutlimitation, trauma (for example; severe injury or surgery), extremeexercise (for example running a marathon), stroke and heart attack,sepsis or other serious infection and endometriosis.

The immunoassays of the invention include immunometric assays employingenzyme detection methods (for example ELISA), fluorescence labelledimmunometric assays, time-resolved fluorescence labelled immunometricassays, chemiluminescent immunometric assays, immunoturbidimetricassays, particulate labelled immunometric assays and immunoradiometricassays and competitive immunoassay methods including labelled antigenand labelled antibody competitive immunoassay methods with a variety oflabel types including radioactive, enzyme, fluorescent, time-resolvedfluorescent and particulate labels. All of said immunoassay methods arewell known in the art, see for example Salgame et al, 1997 and vanNieuwenhuijze et al, 2003.

In one embodiment, said biological sample comprises a body fluid. Forexample, biological samples that may be tested in a method of theinvention include cerebrospinal fluid (CSF), whole blood, blood serum,plasma, menstrual blood, endometrial fluid, urine, saliva, or otherbodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g.as condensed breath, or an extract or purification therefrom, ordilution thereof. Biological samples also include specimens from a livesubject, or taken post-mortem. The samples can be prepared, for examplewhere appropriate diluted or concentrated, and stored in the usualmanner.

In one embodiment, the method of the invention is repeated on multipleoccasions. This embodiment provides the advantage of allowing thedetection results to be monitored over a time period. Such anarrangement will provide the benefit of monitoring or assessing theefficacy of treatment of a disease state. Such monitoring methods of theinvention can be used to monitor onset, progression, stabilisation,amelioration, relapse and/or remission.

Thus, the invention also provides a method of monitoring efficacy of atherapy for a disease state in a subject, suspected of having such adisease, comprising detecting and/or quantifying the biomarker presentin a biological sample from said subject. In monitoring methods, testsamples may be taken on two or more occasions. The method may furthercomprise comparing the level of the biomarker(s) present in the testsample with one or more control(s) and/or with one or more previous testsample(s) taken earlier from the same test subject, e.g. prior tocommencement of therapy, and/or from the same test subject at an earlierstage of therapy. The method may comprise detecting a change in thenature or amount of the biomarker(s) in test samples taken on differentoccasions.

Thus, according to a further aspect of the invention, there is provideda method for monitoring efficacy of therapy for a disease state in ahuman or animal subject, comprising:

-   -   (a) quantifying the amount of the biomarker as defined herein;        and    -   (b) comparing the amount of said biomarker in a test sample with        the amount present in one or more control(s) and/or one or more        previous test sample(s) taken at an earlier time from the same        test subject.

A change in the level of the biomarker in the test sample relative tothe level in a previous test sample taken earlier from the same testsubject may be indicative of a beneficial effect, e.g. stabilisation orimprovement, of said therapy on the disorder or suspected disorder.Furthermore, once treatment has been completed, the method of theinvention may be periodically repeated in order to monitor for therecurrence of a disease.

Methods for monitoring efficacy of a therapy can be used to monitor thetherapeutic effectiveness of existing therapies and new therapies inhuman subjects and in non-human animals (e.g. in animal models). Thesemonitoring methods can be incorporated into screens for new drugsubstances and combinations of substances.

In a further embodiment the monitoring of more rapid changes due to fastacting therapies may be conducted at shorter intervals of hours or days.

According to a further aspect of the invention, there is provided amethod for identifying a biomarker for detecting the presence of adisease state. The term “identifying” as used herein means confirmingthe presence of the biomarker present in the biological sample.Quantifying the amount of the biomarker present in a sample may includedetermining the concentration of the biomarker present in the sample.Identifying and/or quantifying may be performed directly on the sample,or indirectly on an extract therefrom, or on a dilution thereof.

In alternative aspects of the invention, the presence of the biomarkeris assessed by detecting and/or quantifying antibody or fragmentsthereof capable of specific binding to the biomarker that are generatedby the subject's body in response to the biomarker and thus are presentin a biological sample from a subject having a disease state.

Identifying and/or quantifying can be performed by any method suitableto identify the presence and/or amount of a specific protein in abiological sample from a subject or a purification or extract of abiological sample or a dilution thereof. In methods of the invention,quantifying may be performed by measuring the concentration of thebiomarker in the sample or samples. Biological samples that may betested in a method of the invention include those as definedhereinbefore. The samples can be prepared, for example where appropriatediluted or concentrated, and stored in the usual manner.

Identification and/or quantification of biomarkers may be performed bydetection of the biomarker or of a fragment thereof, e.g. a fragmentwith C-terminal truncation, or with N-terminal truncation. Fragments aresuitably greater than 4 amino acids in length, for example 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.It is noted in particular that peptides of the same or related sequenceto that of histone tails are particularly useful fragments of histoneproteins.

The biomarker may be directly detected, e.g. by SELDI or MALDI-TOF.Alternatively, the biomarker may be detected directly or indirectly viainteraction with a ligand or ligands such as an antibody or abiomarker-binding fragment thereof, or other peptide, or ligand, e.g.aptamer, or oligonucleotide, capable of specifically binding thebiomarker. The ligand or binder may possess a detectable label, such asa luminescent, fluorescent or radioactive label, and/or an affinity tag.

For example, detecting and/or quantifying can be performed by one ormore method(s) selected from the group consisting of: SELDI (−TOF),MALDI (−TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Massspec (MS), reverse phase (RP) LC, size permeation (gel filtration), ionexchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques.Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA,USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography(e.g. high pressure liquid chromatography (HPLC) or low pressure liquidchromatography (LPLC)), thin-layer chromatography, NMR (nuclear magneticresonance) spectroscopy could also be used.

Methods of diagnosing or monitoring according to the invention maycomprise analysing a sample by SELDI TOF or MALDI TOF to detect thepresence or level of the biomarker. These methods are also suitable forclinical screening, prognosis, monitoring the results of therapy,identifying subjects most likely to respond to a particular therapeutictreatment, for drug screening and development, and identification of newtargets for drug treatment.

Identifying and/or quantifying the analyte biomarkers may be performedusing an immunological method, involving an antibody, or a fragmentthereof capable of specific binding to the biomarker. Suitableimmunological methods include sandwich immunoassays, such as sandwichELISA, in which the detection of the analyte biomarkers is performedusing two antibodies which recognize different epitopes on a analytebiomarker; radioimmunoassays (RIA), direct, indirect or competitiveenzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA),Fluorescence immunoassays (FIA), western blotting, immunoprecipitationand any particle-based immunoassay (e.g. using gold, silver, or latexparticles, magnetic particles, or Q-dots). Immunological methods may beperformed, for example, in microtitre plate or strip format.

In one embodiment, one or more of the biomarkers may be replaced by amolecule, or a measurable fragment of the molecule, found upstream ordownstream of the biomarker in a biological pathway.

The identification of key biomarkers specific to a disease is central tointegration of diagnostic procedures and therapeutic regimes. Usingpredictive biomarkers appropriate diagnostic tools such as biosensorscan be developed; accordingly, in methods and uses of the invention,identifying and quantifying can be performed using a biosensor,microanalytical system, microengineered system, microseparation system,immunochromatography system or other suitable analytical devices. Thebiosensor may incorporate an immunological method for detection of thebiomarker(s), electrical, thermal, magnetic, optical (e.g. hologram) oracoustic technologies. Using such biosensors, it is possible to detectthe target biomarker(s) at the anticipated concentrations found inbiological samples.

As used herein, the term “biosensor” means anything capable of detectingthe presence of the biomarker. Examples of biosensors are describedherein.

Biosensors according to the invention may comprise a ligand binder orligands, as described herein, capable of specific binding to thebiomarker. Such biosensors are useful in detecting and/or quantifying abiomarker of the invention.

The biomarker(s) of the invention can be detected using a biosensorincorporating technologies based on “smart” holograms, or high frequencyacoustic systems, such systems are particularly amenable to “bar code”or array configurations.

In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), aholographic image is stored in a thin polymer film that is sensitised toreact specifically with the biomarker. On exposure, the biomarker reactswith the polymer leading to an alteration in the image displayed by thehologram. The test result read-out can be a change in the opticalbrightness, image, colour and/or position of the image. For qualitativeand semi-quantitative applications, a sensor hologram can be read byeye, thus removing the need for detection equipment. A simple coloursensor can be used to read the signal when quantitative measurements arerequired. Opacity or colour of the sample does not interfere withoperation of the sensor. The format of the sensor allows multiplexingfor simultaneous detection of several substances. Reversible andirreversible sensors can be designed to meet different requirements, andcontinuous monitoring of a particular biomarker of interest is feasible.

Suitably, biosensors for detection of one or more biomarkers of theinvention combine biomolecular recognition with appropriate means toconvert detection of the presence, or quantitation, of the biomarker inthe sample into a signal. Biosensors can be adapted for “alternate site”diagnostic testing, e.g. in the ward, outsubjects' department, surgery,home, field and workplace.

Biosensors to detect one or more biomarkers of the invention includeacoustic, plasmon resonance, holographic, Bio-Layer Interferometry (BLI)and microengineered sensors. Imprinted recognition elements, thin filmtransistor technology, magnetic acoustic resonator devices and othernovel acousto-electrical systems may be employed in biosensors fordetection of the one or more biomarkers of the invention.

Methods involving identification and/or quantification of one or morebiomarkers of the invention can be performed on bench-top instruments,or can be incorporated onto disposable, diagnostic or monitoringplatforms that can be used in a non-laboratory environment, e.g. in thephysician's office or at the subject's bedside. Suitable biosensors forperforming methods of the invention include “credit” cards with opticalor acoustic readers. Biosensors can be configured to allow the datacollected to be electronically transmitted to the physician forinterpretation and thus can form the basis for e-medicine.

Diagnostic kits for the diagnosis and monitoring of the presence of adisease state are described herein. In one embodiment, the kitsadditionally contain a biosensor capable of identifying and/orquantifying a biomarker. Suitably a kit according to the invention maycontain one or more components selected from the group: a ligand binder,or ligands, specific for the biomarker or a structural/shape mimic ofthe biomarker, one or more controls, one or more reagents and one ormore consumables; optionally together with instructions for use of thekit in accordance with any of the methods defined herein.

The identification of biomarkers for a disease state permits integrationof diagnostic procedures and therapeutic regimes. Detection of abiomarker of the invention can be used to screen subjects prior to theirparticipation in clinical trials. The biomarkers provide the means toindicate therapeutic response, failure to respond, unfavourableside-effect profile, degree of medication compliance and achievement ofadequate serum drug levels. The biomarkers may be used to providewarning of adverse drug response. Biomarkers are useful in developmentof personalized therapies, as assessment of response can be used tofine-tune dosage, minimise the number of prescribed medications, reducethe delay in attaining effective therapy and avoid adverse drugreactions. Thus by monitoring a biomarker of the invention, subject carecan be tailored precisely to match the needs determined by the disorderand the pharmacogenomic profile of the subject, the biomarker can thusbe used to titrate the optimal dose, predict a positive therapeuticresponse and identify those subjects at high risk of severe sideeffects.

Biomarker-based tests provide a first line assessment of ‘new’ subjects,and provide objective measures for accurate and rapid diagnosis, notachievable using the current measures.

Furthermore, diagnostic biomarker tests are useful to identify familymembers or subjects with mild or asymptomatic disease or who may be athigh risk of developing symptomatic disease. This permits initiation ofappropriate therapy, or preventive measures, e.g. managing risk factors.These approaches are recognised to improve outcome and may prevent overtonset of the disorder.

Biomarker monitoring methods, biosensors and kits are also vital assubject monitoring tools, to enable the physician to determine whetherrelapse is due to worsening of the disorder. If pharmacologicaltreatment is assessed to be inadequate, then therapy can be reinstatedor increased; a change in therapy can be given if appropriate. As thebiomarkers are sensitive to the state of the disorder, they provide anindication of the impact of drug therapy.

The invention will now be illustrated with reference to the followingnon-limiting examples.

Example 1

Serum samples were taken from 5 healthy subjects, 3 subjects with coloncancer, 6 subjects with lung cancer and 2 subjects with pancreaticcancer. A commercially available nucleosome preparation produced bydigestion of chromatin extracted from Hela cells, in which the DNA andproteins in the nucleosome are cross-linked for stability, was seriallydiluted in horse serum. A nucleosome preparation in human blood wasprepared according to the method of Holdenrieder (*Holdenrieder et al;2001). These samples and preparations were assayed in duplicate fornucleosome-EZH2 adduct by the method of the invention. Neat commerciallyavailable horse serum produced for use in tissue culture was alsoassayed as a negative control sample containing no nucleosomes ornucleosome adducts.

The ELISA method used a solid phase anti-histone capture antibody thatbinds intact nucleosomes and a biotinylated monoclonal anti-EZH2detection antibody as follows: A solution of anti-histone antibody in0.1M phosphate buffer pH 7.4 was added to microtitre wells (100 μL/well)and incubated overnight at 4° C. to coat the wells with captureantibody. Excess anti-histone antibody was decanted. A solution ofbovine serum albumin (20 g/L) was added to the wells (200 μL/well) andincubated for 30 minutes at room temperature to block excess proteinbinding sites on the wells. Excess bovine serum albumin solution wasdecanted and the wells were washed three times with wash buffer (200μL/well, 0.05M TRIS/HCl buffer pH 7.5 containing 1% Tween 20). Serumsample (10 μL/well) and assay buffer (50 μL/well, 0.05M TRIS/HCl pH 7.5containing 0.9% NaCl, 0.05% sodium deoxycholate and 1% Nonidet P40substitute) were added to the wells incubated overnight at 4° C. Theserum and assay buffer mixture was decanted and the wells were washedthree times with wash buffer (200 μL/well). A solution of biotinylatedanti-EZH2 detection antibody was added (50 μL/well) and incubated for 90minutes at room temperature with mild agitation. Excess detectionantibody was decanted and the wells were again washed three times withwash buffer (200 μL/well). A solution containing a streptavidin-horseradish peroxidase conjugate was added (50 μL/well) and incubated for 30minutes at room temperature with mild agitation. Excess conjugate wasdecanted and the wells were again washed three times with wash buffer(200 μL/well). A coloured substrate solution (100 μL/well, 2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was added andincubated for 20 minutes at room temperature with mild agitation. Theoptical density (OD) of the wells was measured at a wavelength of 405 nmusing a standard microtitre plate reader. A dose response curve ofincreasing colour with increasing nucleosome-EZH2 adduct concentrationwas observed with a low background signal observed in the absence ofnucleosome adduct (horse serum). The positive ELISA signal indicatesthat the EZH2 detected by the ELISA is incorporated within anucleosome-EZH2 adduct comprising both histone protein and EZH2 as (i)the capture antibody binds to histones in the sample and (ii) detectionantibody binds to the EZH2 component of the adduct. The results areshown in FIGS. 1 and 2.

Example 2

Serum samples were taken from 5 healthy subjects, 3 subjects with coloncancer, 6 subjects with lung cancer and 2 subjects with pancreaticcancer. A commercially available nucleosome preparation produced bydigestion of chromatin extracted from Hela cells, was serially dilutedin horse serum. A nucleosome preparation in human blood was preparedaccording to the method of Holdenrieder (*Holdenrieder et al; 2001).These samples and preparations were assayed in duplicate fornucleosome-HMGB1 adduct by the method of the invention. Neat horse serumwas also assayed as a negative control sample containing no nucleosomesor nucleosome adducts.

The ELISA method used a solid phase anti-histone capture antibody thatbinds intact nucleosomes and a biotinylated monoclonal anti-HMGB1detection antibody as follows: A solution of anti-histone antibody in0.1M phosphate buffer pH 7.4 was added to microtitre wells (100 μL/well)and incubated overnight at 4° C. to coat the wells with captureantibody. Excess anti-histone antibody was decanted. A solution ofbovine serum albumin (20 g/L) was added to the wells (200 μL/well) andincubated for 30 minutes at room temperature to block excess proteinbinding sites on the wells. Excess bovine serum albumin solution wasdecanted and the wells were washed three times with wash buffer (200μL/well, 0.05M TRIS/HCl buffer pH 7.5 containing 1% Tween 20). Serumsample (10 μL/well) and assay buffer (50 μL/well, 0.05M TRIS/HCl pH 7.5containing 0.9% NaCl, 0.05% sodium deoxycholate and 1% Nonidet P40substitute) were added to the wells incubated overnight at 4° C. Theserum and assay buffer mixture was decanted and the wells were washedthree times with wash buffer (200 μL/well). A solution of biotinylatedanti-HMGB1 detection antibody was added (50 μL/well) and incubated for90 minutes at room temperature with mild agitation. Excess detectionantibody was decanted and the wells were again washed three times withwash buffer (200 μL/well). A solution containing a streptavidin-horseradish peroxidase conjugate was added (50 μL/well) and incubated for 30minutes at room temperature with mild agitation. Excess conjugate wasdecanted and the wells were again washed three times with wash buffer(200 μL/well). A coloured substrate solution (100 μL/well, 2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was added andincubated for 20 minutes at room temperature with mild agitation. Theoptical density (OD) of the wells was measured at a wavelength of 405 nmusing a standard microtitre plate reader. A dose response curve ofincreasing colour with increasing nucleosome-HMGB1 adduct concentrationwas observed with a low background signal observed in the absence ofnucleosome adduct (horse serum). The positive ELISA signal indicatesthat the HMGB1 detected by the ELISA is incorporated in anucleosome-HMGB1 adduct comprising both histone protein and HMGB1 as (i)the capture antibody binds to histones in the sample and (ii) detectionantibody binds to the HMGB1 component of the adduct. The results areshown in FIGS. 3 and 4.

In a larger experiment, serum samples were taken from 25 patients withcolon cancer, 25 patients with breast cancer and 24 patients with lungcancer as well as samples from 31 healthy subjects. The samples weretested for nucleosome-HMGB1 level and, using the mean healthy resultplus 2 standard deviations in the mean as cut-off, the following resultswere obtained for colon, breast and lung cancer:

-   -   Colon: 76% of cancers were detected (19 of 25 patients) and 90%        specificity (3 false positives from 31 healthy samples);    -   Breast: 96% of cancers were detected (24 of 25 patients) and 90%        specificity (3 false positives from 31 healthy samples); and    -   Lung: 100% of cancers were detected (24 of 24 patients) and 86%        specificity (4 false positives from 28 healthy samples);        where a measured nucleosome-HMGB1 adduct level above the cut-off        level is considered a positive result and a lower level is        considered a negative result. The results are shown in FIG. 5.

The assay for nucleosome-HMGB1 adduct levels was also carried out in thereverse format where the anti-HMGB1 antibody was coated to wells ascapture antibody and the anti-nucleosome antibody was biotinylated andused as detection antibody. This format of the assay also successfullydetected nucleosome-HMGB1 adducts in the positive controls used (OD405nm=1.15) but not in either horse serum or buffer (both OD406 nm=0.13)

Example 3

A nucleosome-PR ELISA assay was carried out using the method of Example1 above except that the biotinylated antibody used was directed to bindthe progesterone receptor (PR). The results are shown in FIG. 6.

Example 4

Serum samples taken from two prostate cancer patients, as well aspositive and negative controls, were assayed using a nucleosome-ARadduct ELISA assay carried out using the method of Example 1 aboveexcept that the biotinylated antibody used was directed to bind theandrogen receptor (AR). The results are shown in FIG. 7.

Example 5

A nucleosome-ERα adduct ELISA assay was carried out using a nucleosomesample prepared by the method of *Holdenrieder et al; 2001 by the methodof Example 1 above except that the biotinylated antibody used wasdirected to bind the alpha form of the estrogen receptor (ERα). Theresults are shown in FIG. 8.

Example 6

A nucleosome sample was prepared by digestion of chromatin extractedfrom MCF7 cells and assayed for nucleosome-ERβ adduct by ELISA. Theassay was carried out by a method similar to that of Example 1 aboveexcept that the assay was performed using a different anti-nucleosomeantibody and an antibody directed to bind the beta form of the estrogenreceptor (ERβ). The assay was carried out in two different formats. Inthe first format the anti-nucleosome antibody was coated on the wellsand the anti-ERβ antibody was biotinylated. In the second format theanti-ERβ antibody was coated on the wells and the anti-nucleosomeantibody was biotinylated. The assay was successful in both formats.Interestingly the assay appeared to perform less well when the MCF7chromatin was cross-linked as is often done in ChIP methods. The resultsare shown in FIG. 9.

Example 7

A nucleosome H2AZ-ERβ adduct ELISA assay was carried out using anucleosome sample prepared by the method of *Holdenrieder et al; 2001 bythe method of Example 6 above where the anti-ERβ antibody was coated onthe wells except that the biotinylated antibody used was directed tobind the histone variant H2AZ such that only the subset ofnucleosome-ERβ adducts containing H2AZ were detected. Using this method,wherein the nucleosome or nucleosome component binder is directed tobind to a particular epigenetic signal structure, it is possible todetect a particular subset of nucleosome adducts containing only thatepigenetic signal. The results are shown in FIG. 10.

Example 8

Serum samples were taken from 12 healthy subjects, 3 subjects with coloncancer, 6 subjects with breast cancer, 3 subjects with lung cancer and 4subjects with pancreatic cancer. A nucleosome preparation in human bloodwas prepared according to the method of Holdenrieder (*Holdenrieder etal; 2001) and was serially diluted in horse serum. These samples andpreparations were assayed in duplicate for nucleosome-ERβ adduct by themethod of the invention. Neat commercially available horse serumproduced for use in tissue culture was also assayed as a negativecontrol sample containing no nucleosomes or nucleosome adducts. Theassay was carried out using the method of Example 1 above except thatthe detection antibody used was directed against the estrogen receptor(ERβ). Using a cut-off calculated as the mean healthy result plus 2standard deviations of the mean; 2 of 3 colon cancer samples, 3 of 6breast cancer samples, 2 of 3 lung cancer samples and 4 of 4 pancreaticcancer samples were found positive for nucleosome-ERβ adduct. Theresults are shown in FIG. 11.

Example 9

A nucleosome-estrogen receptor-steroid estrogen ELISA assay is carriedout using the method of Example 6 above except that the detectionantibody used was directed against steroid estrogen. This assay therefordetected only nucleosome-estrogen receptor adducts that additionallycontained steroid hormone.

Example 10

A nucleosome-estrogen receptor-estrogen adduct ELISA assay is carriedout using a method similar to that of Example above except that theassay is performed in a microtitre plate or tube that is resistant toorganic solvents and, following anti-nucleosome antibody capture ofnucleosome-estrogen receptor adducts on the surface of the well, theliquid contents of the well are decanted and diethylether is added todissolve any steroid present in the captured adduct. The ether istransferred, to another well or tube and dried. The dried extract isredissolved in assay buffer and the estrogen concentration is determinedusing a classical competitive immunoassay method for estrogen analysis.This assay therefore detects only nucleosome-estrogen receptor adductsthat additionally contain estrogen.

Example 11

A nucleosome-retinoic acid receptor-retinoic acid ELISA assay is carriedout using the method of Example 10 above except that the steroidcompetitive immunoassay used was directed against retinoic acid. Thisassay therefor detects only nucleosome-retinoic acid receptor adductsthat additionally contain steroid retinoic acid.

Example 12

Nucleosome adduct assays similar to those described in Examples 1-10 areperformed except that the solid phase coated antibody used was directedagainst 5-methylcytidine. These assays therefor detect onlynucleosome-hormone receptor adducts and nucleosome-hormonereceptor-hormone complex adducts which are additionally associated withmethylated DNA.

REFERENCES

-   Allen et al, A simple method for estimating global DNA methylation    using bisulfite PCR of repetitive DNA elements. Nucleic Acids    Research: 32(3) e38DOI: 10.1093/nar/gnh032, 2004-   Bawden et al, Detection of histone modification in cell-free    nucleosomes. WO 2005/019826, 2005-   Cao et al, Role of Histone H3 Lysine 27 Methylation in    Polycomb-Group Silencing SCIENCE 298, 1039-1043, 2002-   Dai et al, Detection of Post-translational Modifications on Native    Intact Nucleosomes by ELISA. http://www.jove.com/details.php?id=2593    doi: 10.3791/2593. J Vis Exp. 50 (2011).-   Esteller, Cancer epigenomics: DNA methylomes and    histone-modification maps Nature Reviews Genetics: 8, 286-298, 2007-   Feinberg and Vogelstein, Hypomethylation distinguishes genes of some    human cancers from their normal counterparts. Nature: 301, 89-92,    1983-   Fullgrabe et al, Histone onco-modifications. Oncogene: 30,    3391-3403, 2011-   Gerlitz et al, The dynamics of HMG protein-chromatin interactions in    living cells. Biochem Cell Biol, 87, 127-137, 2009-   Grutzmann et al, Sensitive Detection of Colorectal Cancer in    Peripheral Blood by Septin 9 DNA Methylation Assay. PLoS ONE 3(11):    e3759. doi:10.1371/journal.pone.0003759, 2008-   Herranz and Esteller, DNA methylation and histone modifications in    subjects with cancer: potential prognostic and therapeutic targets.    Methods Mol Biol. 361:25-62, 2007-   Hervouet et al, Disruption of Dnmt1/PCNA/UHRF1 Interactions Promotes    Tumorigenesis from Human and Mice Glial Cells PLoS ONE 5(6): e11333.    doi:10.1371/journal.pone.0011333, 2010-   Holdenrieder et al, Nucleosomes in serum of subjects with benign and    malignant diseases. Int. J. Cancer (Pred. Oncol.): 95, 114-120, 2001-   Holdenrieder et al, Nucleosomes in Serum as a Marker for Cell Death.    Clin Chem Lab Med; 39(7), 596-605, 2001-   Holdenrieder et al, Cell-Free DNA in Serum and Plasma: Comparison of    ELISA and Quantitative PCR. Clinical Chemistry: 51(8), 1544-1546,    2005-   Holdenrieder and Stieber, Clinical use of circulating nucleosomes.    Critical Reviews in Clinical Laboratory Sciences; 46(1): 1-24, 2009-   Ricke and Bielinsky, Easy detection of chromatin binding proteins by    the histone association assay. Biol Proced Online; 7(1), 60-69, 2005-   Rodriguez-Paredes and Esteller, Cancer epigenetics reaches    mainstream oncology. Nature Medicine: 17(3), 330-339, 2011-   Salgame et al, An ELISA for detection of apoptosis. Nucleic Acids    Research, 25(3), 680-681, 1997-   Sims et al, HMGB1 and RAGE in inflammation and cancer. Annu. Rev.    Immunol. 28, 367-388, 2010-   Stoetzer et al, Circulating nucleosomes and biomarkers of    immunogenic cell death as predictive and prognostic markers in    cancer patients undergoing cytotoxic therapy. Expert Opin Biol Ther.    12(Suppl. 1): S217-S224, 2012-   Tang et al, High-mobility Group Box 1 [HMGB1] and Cancer. Biochim    Biophys Acta. 1799(1-2) 131, 2010-   Urbonaviciute et al, Induction of inflammatory and immune responses    by HMGB1—nucleosome complexes: implications for the pathogenesis of    SLE. J Exp Med, 205(13), 3007-3018, 2008-   Urbonaviciute and Voll, High-mobility group box 1 represents a    potential marker of disease and novel therapeutic target in systemic    lupus erythematosus. J Internal Medicine, 270, 309-318, 2011-   van Nieuwenhuijze et al, Time between onset of apoptosis and release    of nucleosomes from apoptotic cells: putative implications for    sysytemic lupus erythematosus. Ann Rheum Dis; 62: 10-14, 2003-   Yoshida and Shimura, Isolation of nonhistone chromosomal protein    from calf thymus. Biochimica et Biophysica Acta (BBA)—Protein    Structure; 263(3), 690-695, 1972

1. Use of a nucleosome-protein adduct as a biomarker in blood for thediagnosis of cancer, autoimmune disease or inflammatory disease.
 2. Useas defined in claim 1, wherein the biomarker is used for the diagnosisof cancer.
 3. Use as defined in claim 1 or claim 2, wherein thenucleosome-protein adduct includes a chromatin modifying enzyme, anuclear receptor or a hormone.
 4. Use as defined in claim 2, wherein thenucleosome-protein adduct includes a High Mobility Group Protein.
 5. Useas defined in claim 3, wherein the chromatin modifying enzyme is ahistone acetylation, deacetylation, methylation, demethylationphosphorylation, dephosphorylation ubiquitination, deubiquitinationsumoylation, desumoylation or DNA methyltransferase enzyme.
 6. Use asdefined in claim 3, wherein the nuclear receptor is the estrogenreceptor, androgen receptor, progesterone receptor, thyroid hormonereceptor, glucocorticoid receptor or retinoic acid receptor.
 7. Use asdefined in claim 3, wherein the hormone is a thyroid hormone, aglucocorticoid hormone or a steroid hormone including an estrogen, anandrogen, a progestogen, a corticosteroid or retinoic acid.
 8. A methodfor detecting the presence of a nucleosome-protein adduct in a samplewhich comprises the steps of: (i) contacting the sample with a firstbinding agent which binds to nucleosomes or a component thereof; (ii)contacting the nucleosomes or sample with a second binding agent whichbinds to a protein adducted to a nucleosome; (iii) detecting orquantifying the binding of said second binding agent to the adductedprotein in the sample; and (iv) using the presence or degree of suchbinding as a measure of the presence of nucleosome adducts in thesample.
 9. A method for detecting the presence of a nucleosome adduct ina sample which comprises the steps of: (i) contacting a sample with afirst binding agent which binds to a protein adducted to a nucleosome;(ii) contacting the nucleosomes or sample with a second binding agentwhich binds to nucleosomes or a component thereof; (iii) detecting orquantifying the binding of said second binding agent to nucleosomes or acomponent thereof in the sample; and (iv) using the presence or degreeof such binding as a measure of the presence of nucleosome adducts inthe sample.
 10. The method as defined in claim 8 or claim 9, wherein thenucleosome adduct includes a pro-inflammatory protein, a High MobilityGroup Protein, a polycomb protein, a chromatin modifying enzyme, anuclear receptor or a hormone.
 11. The method as defined in claim 10,wherein the High Mobility Group Protein is HMGB1.
 12. The method asdefined in claim 10, wherein the chromatin modifying enzyme is EZH2. 13.The method as defined in claim 10, wherein the hormone receptor is theestrogen receptor, androgen receptor, progesterone receptor, thyroidhormone receptor or retinoic acid receptor.
 14. The method as defined inclaim 10, wherein the hormone is a thyroid hormone, a glucocorticoidhormone or a steroid hormone including an estrogen, an androgen, aprogestogen or retinoic acid.
 15. The method as defined in any one ofclaims 8 to 14, wherein the nucleosome or nucleosome component binder isdirected to bind to a particular epigenetic signal structure such thatonly a particular subset of nucleosome adducts containing saidepigenetic signal structure are detected.
 16. The method as defined inany one of claims 8 to 15, wherein the binding agent is an antibody, anantibody fragment or an aptamer.
 17. The method as defined in any one ofclaims 8 to 16, wherein the sample is a biological fluid.
 18. A methodas defined in any one of claims 8 to 17, wherein the sample is blood orserum or plasma.
 19. A method for detecting the presence of anucleosome-protein adduct as defined in any one of claims 8 to 18, in acell which comprises the steps of: (i) isolating chromatin from a cell;(ii) digesting, sonicating or otherwise breaking down the chromatin toform mono-nucleosomes and/or oligo-nucleosomes; and (iii) detecting ormeasuring the presence of the nucleosome adduct as defined in a methodof any one of claims 8 to
 18. 20. A method for detecting or diagnosing adisease status in an animal or a human subject which comprises the stepsof: (i) detecting or measuring a nucleosome adduct in a body fluid of asubject as defined in either of the methods of claim 8 or claim 9; and(ii) using the nucleosome adduct level detected to identify the diseasestatus of the subject.
 21. A method for assessment of an animal or ahuman subject for suitability for a medical treatment which comprisesthe steps of: (i) detecting or measuring a nucleosome adduct in a bodyfluid of the subject as defined in either of the methods of claim 8 orclaim 9; and (ii) using the nucleosome adduct level detected as aparameter for selection of a suitable treatment for the subject.
 22. Amethod for monitoring a treatment of an animal or a human subject whichcomprises the steps of: (i) detecting or measuring a nucleosome adductin a body fluid of the subject as defined in either of the methods ofclaim 8 or claim 9; (ii) repeating the detection or measurement of anucleosome adduct in a body fluid of the subject on one or moreoccasions; (iii) using any changes in the nucleosome adduct leveldetected as a parameter for any changes in the condition of the subject.23. A method as defined in any one of claims 20 to 22, wherein thenucleosome adduct is detected or measured as one of a panel ofmeasurements.
 24. A method as defined in any one of claims 20 to 23, foruse in subjects with actual or suspected cancer, benign tumours,inflammatory disease, autoimmune disease, endometriosis, infectiousdisease, sepsis, stroke or myocardial infarction.
 25. A method asdefined in any one of claims 8 to 24 for use in detectinghormone-hormone receptor-nucleosome complex adducts.
 26. The method asdefined in claim 25, wherein the hormone-hormone receptor-nucleosomecomplex adducts comprise a thyroxine-thyroid hormone receptor-nucleosomecomplex adduct, a triiodothyronine-thyroid hormone receptor-nucleosomecomplex adduct, a retinoic acid-retinoic acid receptor-nucleosomecomplex adduct, an androgen-androgen receptor-nucleosome complex adductor an estrogen-estrogen receptor-nucleosome complex adduct.
 27. Themethod as defined in claim 25 or claim 26, which comprises an antibodyor other binder directed to bind to the hormone.
 28. The method asdefined in claim 25 or claim 26, which comprises the step of extractingthe hormone from an antibody captured hormone-hormonereceptor-nucleosome complex adduct followed by a quantification step.29. A method for identifying a nucleosome adduct biomarker for detectingor diagnosing a disease status in an animal or a human subject whichcomprises the steps of: (i) detecting or measuring a nucleosome adductin a body fluid of the subject as defined in either of the methods ofclaim 8 or claim 9; (ii) detecting or measuring a nucleosome adduct in abody fluid of a healthy subject or a control subject; and (iii) usingthe difference between the levels detected in diseased and controlsubjects to identify whether a nucleosome adduct is useful as abiomarker for the disease status.
 30. A biomarker identified by themethod as defined in claim
 29. 31. A kit for the detection of anucleosome adduct which comprises a ligand or binder specific for theprotein in the adduct or component part thereof, or a structural/shapemimic of the protein in the adduct or component part thereof, togetherwith instructions for use of the kit in accordance with any one of themethods defined in claims 3 to 28.